In the U.S., metastatic colon carcinoma is second only to lung cancer as the cause of death from malignancy in the United States, accounting for 60,000 fatalities a year. 80 percent of the patients who died of colon cancer have metastases in the liver, and half of them have only liver metastases at the time of death. Current treatments include surgery and chemotherapy, with a mean survival time of only 37 months. Using an orthotopic murine model for pre-established hepatic colon cancer, we have shown that intratumoral injection of an adenoviral vector expressing murine interleukin-12 (Adv.mIL-12) led to tumor regression and prolonged survival of treated mice. However, the anti-tumor immunity induced by this type of gene therapy was mediated primarily by natural killer (NK) cell. In order to also induce a tumor specific T cell response as well, we co-administered an agonistic monoclonal antibody against murine 4-1BB, a co-stimulatory molecule on activated T cells, to induce CD8+ T cell activation and proliferation. The combination treatment resulted in the induction of a strong anti-tumoral immunity and 80 percent long-term survival in the tumor-bearing animals (5x5 m2), even with an eighteen-fold reduction of the Adv.RSV-mIL-12 dose. However, the combination treatment can only induce an NK and CD8+ T cells mediated anti-tumor responses and is less effective for large tumors (lOx10 mm2). In order to achieve persistent anti-tumor immunity and improve therapeutic effect when treating large tumors, the activation of CD4+ T helper cells will be incorporated into the combination therapy via agonistic antibodies against the co-stimulatory molecule OX40, an activation molecule on activated CD4 T helper cells, or CD40, an activation molecule on activated antigen presenting cells (APC). Three specific aims will be pursued: 1) To determine whether intra-tumoral IL-12 gene delivery in conjunction with agonistic antibodies against 4-1BB plus OX40 and/or CD40 can achieve better therapeutic effect for large tumors and generate persistent anti-tumor immune responses to prevent residual and metastatic tumors. 2) To examine the role of CD4 T cells in the accentuated development of tumor specific CD8 memory T cells in combination treated animals. 3) To investigate whether the CD4O and OX 40 activated immune cells can overcome the immune tolerance in mice with a large tumor burden. Successful completion of these studies will result in a better understanding of the interaction of APC, CD4, and CD8 cells in the establishment of anti-tumor memory CD8 T cells and immune suppression/tolerance mediated by a large tumor burden. These therapeutic strategies will be utilized as the scientific foundation for active immune modulation of patients with metastatic colorectal cancer.